Envenomations by the brown recluse spider, Loxosceles reclusa, are a significant source of morbidity in endemic regions of the United States, and misdiagnoses are common. A survey of physicians in the endemic area has shown the economic viability of an accurate diagnostic test for these spider bites. Development and testing of an optimized Loxosceles venom assay will present significant challenges. Unlike the routine construction of ELISAs dedicated to the detection of a single protein, this ELISA will detect venom containing multiple proteins, including a unique physiologically active protein- sphingomyelinase D (SMD) abundantly present in the venom. In preliminary research, our polyclonal assay has shown good sensitivity and good in-vitro specificity. Our research shows that identifiable amounts of venom in clinical envenomations are present for at least seven days. In rabbits, our polyclonal assay allows identification of venom on the surface for as long as two weeks. The limits of sensitivity, in-vivo specificity, and the duration of detection are unknown. Phase I should allow determination of the smallest amount of venom detectable as well as the clinical time limits for in-vivo duration of sensitivity and specificity of the assay. Monoclonal antibodies will be isotyped and quantities raised in a bioreactor to perform checkerboard analysis. This analysis will allow determination of an optimal combination of L. reclusa monoclonal and polyclonal antibodies. A kit assay will result from Phase I, allowing multi-site testing in Phase II. These clinical studies will allow development of a lateral flow assay or microtiter plate assay, with the goal of FDA device approval and marketing. Clinical application of an optimized assay would save the morbidity and expense due to inappropriate diagnosis and treatment of various skin conditions with presentations similar to Loxosceles envenomations. Techniques used in the successful detection of this spider venom are directly applicable to bites from S. American Loxosceles species, responsible for additional deaths each year. The swab venom assay technique could be applicable to envenomations from numerous species PUBLIC HEALTH RELEVANCE: Bites of the brown recluse spider, Loxosceles reclusa, cause considerable morbidity and occasional mortality in the Midwest. Many lesions can appear similar and mimic spider bites, including bacterial and fungal skin infections as well as skin cancer. The goal of this project is to develop a commercially viable test for brown recluse spider bites using a painless and simple swab test for the spider venom on the surface of the skin.